Shortened purification process for the production of capsular streptococcus pneumoniae polysaccharides

ABSTRACT

A shortened process for producing a solution containing substantially purified capsular polysaccharides from a cellular  Streptococcus pneumoniae  lysate broth is described. Ultrafiltering and diafiltering a clarified  S. pneumoniae  lysate followed by pH adjustment to less than 4.5, preferably about 3.5, precipitated at least 98% of the protein in the solution without seriously affecting polysaccharide yield. Furthermore, following ultrafiltration and diafiltration and acidification to a pH of less than 4.5, filtration using activated carbon precipitated at least 90% of remaining protein without seriously affecting polysaccharide yield. Exemplary, non-limiting S. pneumoniae serotypes that can be purified using the shortened process of the invention are 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the  Streptococcus pneumoniae  cells are lysed using deoxycholate sodium (DOC), while in another embodiment the lytic agent is a non-animal derived lytic agent such as N-lauryl sarcosine sodium (NLS).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/163,863, filed Jan. 24, 2014, which is a divisional application ofU.S. application Ser. No. 12/052,525, filed Mar. 20, 2008, which claimsthe benefit of U.S. Provisional Application No. 60/896,616, filed Mar.23, 2007, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods for removing excess soluble proteinand other impurities from cellular lysates of Streptococcus pneumoniae(S. pneumoniae) serotypes used in the production of purifiedpneumococcal polysaccharides.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae are Gram-positive, lancet shaped cocci that areusually seen in pairs (diplococci), but also in short chains or assingle cells. They grow readily on blood agar plates with glisteningcolonies and display alpha hemolysis unless grown anaerobically wherethey show beta hemolysis. They are sensitive to bile salts that canbreak down the cell wall with the presence of the cells' own enzyme,autolysin. The organism is an aerotolerant anaerobe and is fastidious inthat it has complex nutritional requirements.

The cells of most pneumococcal serotypes have a capsule which is apolysaccharide coating surrounding each cell. This capsule is adeterminant of virulence in humans because it interferes withphagocytosis by preventing antibodies from attaching to the bacterialcells. There are currently 90 capsular serotypes identified, with 23serotypes responsible for about 90% of invasive disease. As a vaccinethe polysaccharide can confer a reasonable degree of immunity to S.pneumoniae in individuals with developed or unimpaired immune systems.However, when the polysaccharide is conjugated with a high molecularweight protein such as CRM₁₉₇ and formulated into a vaccine containingconjugates of multiple serotypes, such conjugate vaccines allow for animmune response in infants and elderly who are also most at risk forpneumococcal infections.

The capsular polysaccharide for each S. pneumoniae serotype utilized forvaccine products is produced by growing the organism in liquid medium.The population of the organism is often scaled up from a seed vial toseed bottles and passed through one or more seed fermentors ofincreasing volume until production scale fermentation volumes arereached. The end of the growth cycle can be determined by one of severalmeans, at which point the cells are lysed through the addition of adetergent or other reagent which aids in the cell wall breakdown andrelease of autolysin which causes cellular lysis when the cells reachstationary phase. The broth is then harvested for downstream(purification) processing. The major contaminants are cellular proteins,nucleic acids, C-polysaccharide and medium components.

For most of the serotypes for the currently marketed 7-valentpneumococcal conjugate (7vPnC) vaccine (Prevnar®), as well as the newlydeveloped 13-valent pneumococcal conjugate (13vPnC) vaccine, the currentpurification process requires sixteen steps involving many expensive,labor intensive and technologically demanding operations, such aschromatography and multiple membrane separations. Previous attempts atimproving purification processes for S. Pneumoniae polysaccharides haveincluded, for example, pH manipulation during fermentation and recovery(see U.S. Patent App. Pub. No. 2006/0228381) and solvent and detergentprecipitation. However, the removal of impurities in these processes isstill spread over many labor intensive and costly steps. Protein levelis the most problematic specification to meet due to the physical andchemical properties of the soluble proteins.

Thus, there is a need for a simplified purification process to reducethe soluble protein levels in S. pneumoniae lysates and eliminateinefficiencies of the current purification process to producesubstantially purified capsular polysaccharides suitable forincorporation into pneumococcal conjugate vaccines.

SUMMARY OF THE INVENTION

The present invention relates to a process for producing a solutioncontaining substantially purified capsular polysaccharides from aStreptococcus pneumoniae cell lysate. This process comprises the stepsof:

(a) providing a fermentation broth comprising bacterial cells thatproduce a selected Streptococcus pneumoniae serotype;

(b) lysing the bacterial cells in step (a) with a lytic agent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) to remove low molecular weight impurities and increasepolysaccharide concentration, thereby producing a retentate;

(e) lowering the pH of the retentate of step (d) to less than 4.5 toprecipitate protein and nucleic acids, thereby forming an acidifiedretentate solution;

(f) holding the acidified retentate solution formed in step (e) for atime sufficient to allow settling of the precipitate, followed byfiltration or centrifugation of the acidified retentate solution,thereby producing a clarified polysaccharide solution;

(g) filtering the clarified polysaccharide solution of step (f) throughan activated carbon filter;

(h) ultrafiltering and diafiltering the filtered solution produced bystep (g), thereby producing a concentrated purified polysaccharidesolution; and

(i) filtering the concentrated purified polysaccharide solution producedby step (h) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides is produced. Exemplary, non-limiting S. pneumoniaeserotypes selected for this embodiment of the invention are 1, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In a particular embodiment, thepH of step (e) is lowered to about 3.5. In another embodiment, thediafiltration of step (h) comprises a pH adjustment to between about 5.5to about 7.5. In another embodiment, the diafiltration of step (h)comprises a pH adjustment to between about 7.0 to about 7.5. In anotherembodiment, the diafiltration of step (h) comprises a pH adjustment toabout 7.4. In still another embodiment, step (e) removes at least 98% ofprotein from the retentate of step (d). In another embodiment, step (g)removes at least 90% of the protein from the clarified polysaccharidesolution of step (f). In another embodiment, the activated carbon filterof step (g) comprises wood-based phosphoric acid-activated carbon. Inanother embodiment, step (f) comprises holding the acidified retentatesolution formed in step (e) for at least 2 hours. In still anotherembodiment, the lytic agent of step (b) is deoxycholate sodium (DOC). Inanother embodiment, the lytic agent of step (b) is a non-animal derivedlytic agent. In still another embodment, the lytic agent of step (b) isthe non-animal derived lytic agent N-lauryl sarcosine sodium (NLS).

The present invention also relates to a process for producing a solutioncontaining substantially purified capsular polysaccharides from aStreptococcus pneumoniae cell lysate comprising serotype 1, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19F, or 23F. This process comprises the steps of:

(a) providing a fermentation broth comprising bacterial cells thatproduce Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19F, or 23F;

(b) lysing the bacterial cells in step (a) with a lytic agent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) at room temperature at neutral pH in salt free media to remove lowmolecular weight impurities and increase polysaccharide concentration,thereby producing a salt free retentate;

(e) lowering the pH of the salt free retentate of step (d) to less than4.5 to precipitate protein and nucleic acids, thereby forming anacidified retentate solution;

(f) holding the acidified retentate solution formed in step (e) for atleast 2 hours at room temperature to allow settling of the precipitate,followed by filtration or centrifugation of the acidified retentatesolution, thereby producing a clarified polysaccharide solution;

(g) filtering the clarified polysaccharide solution of step (f) throughan activated carbon filter;

(h) ultrafiltering and diafiltering the filtered solution produced bystep (g), thereby producing a concentrated purified polysaccharidesolution; and

(i) filtering the concentrated purified polysaccharide solution producedby step (h) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides comprising serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19F, or 23F is produced. In a particular embodiment, the pH of step (e)is lowered to about 3.5. In another embodiment, the diafiltration ofstep (h) comprises a pH adjustment to between about 5.5 to about 7.5. Inanother embodiment, the diafiltration of step (h) comprises a pHadjustment to between about 7.0 to about 7.5. In another embodiment, thediafiltration of step (h) comprises a pH adjustment to about 7.4. Instill another embodiment, step (e) removes at least 98% of protein fromthe salt free retentate of step (d). In another embodiment, step (g)removes at least 90% of the protein from the clarified polysaccharidesolution of step (f). In another embodiment, the activated carbon filterof step (g) comprises wood-based phosphoric acid-activated carbon. Instill another embodiment, the lytic agent of step (b) is deoxycholatesodium (DOC). In another embodiment, the lytic agent of step (b) is anon-animal derived lytic agent. In still another embodment, the lyticagent of step (b) is the non-animal derived lytic agent N-laurylsarcosine sodium (NLS).

The present invention also relates to a process for producing a solutioncontaining substantially purified capsular polysaccharides from aStreptococcus pneumoniae cell lysate comprising serotype 19A. Thisprocess comprises the steps of:

(a) providing a fermentation broth comprising bacterial cells thatproduce Streptococcus pneumoniae serotype 19A;

(b) lysing the bacterial cells in step (a) with a lytic agent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) at about 4° C. at a pH of about 6 in sodium phosphate buffer toremove low molecular weight impurities and increase polysaccharideconcentration, thereby producing a retentate;

(e) lowering the pH of the retentate of step (d) to less than 4.5 toprecipitate protein and nucleic acids, thereby forming an acidifiedretentate solution;

(f) holding the acidified retentate solution formed in step (e) for atleast 2 hours at about 4° C. to allow settling of the precipitate,followed by filtration or centrifugation of the acidified retentatesolution, thereby producing a clarified polysaccharide solution;

(g) adjusting the pH of the clarified polysaccharide solution of step(f) to about 6, thereby producing a pH-adjusted clarified polysaccharidesolution;

(h) filtering the pH-adjusted clarified polysaccharide solution of step(g) through an activated carbon filter;

(i) ultrafiltering and diafiltering the filtered solution produced bystep (h), thereby producing a concentrated purified polysaccharidesolution; and

(j) filtering the concentrated purified polysaccharide solution producedby step (i) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides comprising serotype 19A is produced. In a particularembodiment, the pH of step (e) is lowered to about 3.5. In anotherembodiment, the diafiltration of step (i) comprises a pH adjustment tobetween about 5.5 to about 7.5. In another embodiment, the diafiltrationof step (i) comprises a pH adjustment to between about 7.0 to about 7.5.In another embodiment, the diafiltration of step (i) comprises a pHadjustment to about 7.4. In still another embodiment, step (e) removesat least 98% of protein from the retentate of step (d). In anotherembodiment, step (h) removes at least 90% of the protein from thepH-adjusted clarified polysaccharide solution of step (g). In anotherembodiment, the activated carbon filter of step (h) comprises wood-basedphosphoric acid-activated carbon. In another embodiment, the sodiumphosphate buffer of step (d) is 25 mM sodium phosphate. In still anotherembodiment, the lytic agent of step (b) is deoxycholate sodium (DOC). Inanother embodiment, the lytic agent of step (b) is a non-animal derivedlytic agent. In still another embodment, the lytic agent of step (b) isthe non-animal derived lytic agent N-lauryl sarcosine sodium (NLS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows average in-process polysaccharide (PS) yield, protein/PSratio, and nucleic acid (NA)/PS ratio for serotype 5 using the shortenedpurification process of the invention. Results are shown for eachpurification step.

FIG. 2A shows the NMR spectrum for serotype 4 PS from the currentpurification process and FIG. 2B shows the NMR spectrum for serotype 4PS from the shortened purification process. No significant differencesbetween the two spectra were observed. The second peak from the right inboth spectra was pyruvate, and the pyruvate group peak height wascomparable in both spectra.

FIG. 3 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 4 using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 4 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 19A using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 5 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 7F using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 6 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 6B using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 7 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 6A using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 8 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 1 using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 9 shows average in-process PS yield, protein/PS ratio, and NA/PSratio for serotype 14 using the shortened purification process of theinvention. Results are shown for each purification step.

FIG. 10 shows a comparison of PS in-process yields for serotypes 1, 4,5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F purified using the shortenedpurification process of the invention.

FIG. 11 shows a comparison of protein/PS ratios for serotypes 1, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F purified using the shortenedpurification process of the invention. Results are compared for eachpurification step.

FIG. 12 shows a comparison of NA/PS ratios for serotypes 1, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, and 19F purified using the shortenedpurification process of the invention. Results are compared for eachpurification step.

FIG. 13 shows protein removal efficiency attributable to theacidification step of the shortened purification process of theinvention. The difference in protein concentration (SDS-PAGE) before andafter acidification is plotted against initial protein concentrationbefore acidification for batches of serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, and 19F. The protein concentration difference divided bythe initial protein concentration reflected the protein removal rate bythe acidification step.

FIG. 14 shows protein removal efficiency attributable to the carbonadsorption step of the shortened purification process of the invention.The amount of protein removed (adsorbed on carbon) was plotted againstinitial protein loading amounts for batches of serotypes 1, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, and 19F. The amount of protein removed dividedby the initial protein loading amounts reflected the protein removalrate by the carbon adsorption step.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a shortened purification process toreduce the soluble protein levels in cellular Streptococcus pneumoniaelysates to produce substantially purified capsular polysaccharidessuitable for incorporation into pneumococcal conjugate vaccines. Formost of the serotypes for the currently marketed 7-valent pneumococcalconjugate (7vPnC) vaccine (Prevnar®), as well as the newly developed13-valent pneumococcal conjugate (13vPnC) vaccine, the currentpolysaccharide purification process requires up to sixteen steps. Thesesteps involve many expensive, labor-intensive, and technologicallydemanding operations, such as chromatography and multiple membraneseparations. The process of the present invention eliminates up to eightof these steps while accomplishing the same purification and eliminatesthe need for chromatography. Thus, the present invention relates to amore efficient purification process that is less expensive, takes lesstime, and involves fewer steps.

The shortened purification process of the present invention relates tothe discovery that ultrafiltering and diafiltering a clarified cellularS. pneumoniae lysate broth, followed by acidification of theconcentrated lysate broth to a pH of less than 4.5, preferably about3.5, precipitates at least 98% of the protein in the solution withoutseriously affecting polysaccharide yield. By ultrafiltering anddiafiltering the lysed and clarified fermentation broth beforeacidification to a pH of less than 4.5, preferably around 3.5, “saltingin” effects of proteins are eliminated and the fraction of protein thatis “salted out” is increased. “Salting in” refers to increasedsolubility of proteins while “salting out” refers to precipitation ofproteins in solution as they reach their isoelectric points. Theultrafiltration and diafiltration step also prevents the foamingobserved when sodium-carbonate treated broth undergoes acidificationeven to a pH of 5.0 (see U.S. Patent App. Pub. No. 2006/0228381). Thus,ultrafiltration and diafiltration of the clarified lysate broth makes itpossible to use any low molecular weight pH titrant such as sodiumcarbonate during S. pneumoniae serotype fermentation and preventsfoaming of the clarified lysate broth when acidified to a pH of lessthan 4.5.

“Clarified lysate broth” refers to a lysate broth that has undergonecentrifugation or filtration to remove cell debris.

“Diafiltering,” “diafiltration,” “DF,” and like terms refer to, forexample, using semi-permeable membranes with appropriate physical andchemical properties to remove small molecules from a solution.

“Ultrafiltering,” “ultrafiltration,” “UF,” and like terms refer to, forexample, using semi-permeable membranes with appropriate physical andchemical properties to discriminate between molecules in a solution andconcentrate like molecules into a smaller volume of solution.

Within the methods of the present invention, ultrafiltration anddiafiltration typically comprise “cross-flow” or “tangential-flow”filtration in order to avoid clogging of the filter membranes. In“cross-flow” filtration, the solution to be filtered is passed acrossthe surface of the membrane. Materials which pass through the membraneare referred to as the permeate. The materials which do not pass throughthe membrane are referred to as the retentate. The retentate is recycledto a feed reservoir to be refiltered.

As used herein, any acid may be used to lower the pH of theultrafiltered and diafiltered lysate broth so long as a pH of less than4.5, particularly about 3.5, is achieved. Accordingly, both organic andmineral acids may be used within the methods of the invention. As usedherein, the term “mineral acid” refers to an acid derived from inorganicmineral by chemical reaction as opposed to organic acids. Exemplary,non-limiting examples of mineral acids that may be used within themethods of the present invention include hydrochloric acid, nitric acid,phosphoric acid, and sulphuric acid. In particular embodiments, the pHof the concentrated lysate broth is lowered to less than 4.4, 4.3, 4.2,4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, or 3.0. In otherembodiments, the pH of the concentrated lysate broth is lowered to about4.4, about 4.3, about 4.2, about 4.1, about 4.0, about 3.9, about 3.8,about 3.7, about 3.6, about 3.5, about 3.4, about 3.3, about 3.2, about3.1, about 3.0, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5,about 2.4, about 2.3, about 2.2, about 2.1, or about 2.0.

The shortened purification process of the present invention also relatesto the discovery that, in combination with the concentration and low pHsteps described above, filtration using activated carbon precipitates atleast 90% of remaining protein without seriously affectingpolysaccharide yield. In particular embodiments, carbon filtration usingcarbon derived from sawdust or other wood products and activated withphosphoric acid was found to be more effective at reducing or removingprotein impurities than carbons used within current carbon filtrationmethods.

Accordingly, the present invention relates to a process for producing asolution containing substantially purified capsular polysaccharides froma Streptococcus pneumoniae cell lysate comprising the steps of:

(a) providing a fermentation broth comprising bacterial cells thatproduce a selected Streptococcus pneumoniae serotype;

(b) lysing the bacterial cells in step (a) with a lytic agent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) to remove low molecular weight impurities and increasepolysaccharide concentration, thereby producing a retentate;

(e) lowering the pH of the retentate of step (d) to less than 4.5,particularly about 3.5, to precipitate protein and nucleic acids,thereby forming an acidified retentate solution;

(f) holding the acidified retentate solution formed in step (e) for atime sufficient to allow settling of the precipitate, particularly forat least 2 hours with or without agitation, followed by filtration orcentrifugation of the acidified retentate solution, thereby producing aclarified polysaccharide solution;

(g) filtering the clarified polysaccharide solution of step (f) throughan activated carbon filter, particularly an activated carbon filtercomprising wood-based phosphoric acid-activated carbon;

(h) ultrafiltering and diafiltering the filtered solution produced bystep (g), thereby producing a concentrated purified polysaccharidesolution; and

(i) filtering the concentrated purified polysaccharide solution producedby step (h) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides is produced. The sterile filtration of step (i) isuseful to remove bacteria and particles from the concentrated purifiedpolysaccharide solution. Exemplary, non-limiting S. pneumoniae serotypesselected for this embodiment of the invention are 1, 4, 5, 6A, 6B, 7F,9V, 14, 18C, 19A, 19F, and 23F. In a particular embodiment, step (e)removes at least 98% of protein from the retentate of step (d). Inanother embodiment, step (g) removes at least 90% of the protein fromthe clarified polysaccharide solution of step (f). In anotherembodiment, the diafiltration of step (h) comprises a pH adjustment tobetween about 5.5 to about 7.5. For improved stability of thesubstantially purified capsular polysaccharide during long-term storage,however, the diafiltration of step (h) comprises a pH adjustment tobetween about 7.0 to about 7.5, and more particularly to about 7.4.

As used herein, the term “substantially purified capsularpolysaccharide-containing lysate” or “solution containing substantiallypurified capsular polysaccharides” refers to a cellular Streptococcuspneumoniae lysate or solution from which protein has been removed suchthat the percent ratio of protein to polysaccharide (protein/PS) is lessthan 10%, less than 9%, less than 8%, less than 7%, less than 6%, lessthan 5%, less than 4%, less than 3%, less than 2%, or less than 1% andthe percent ratio of nucleic acid to polysaccharide (NA/PS) is less than5%, less than 4%, less than 3%, less than 2%, or less than 1%. Inparticular embodiments, the percent ratios of protein/PS and NA/PS forsubstantially purified capsular polysaccharide-containing lysates orsolutions comprising specific serotypes are as follows: for serotype 1the ratio of protein/PS is less than 2% and the ratio of NA/PS is lessthan 2%, for serotype 4 the ratio of protein/PS is less than 3% and theratio of NA/PS is less than 2%, for serotype 5 the ratio of protein/PSis less than or equal to 7.5% and the ratio of NA/PS is less than orequal to 2%, for serotype 6A the ratio of protein/PS is less than 2% andthe ratio of NA/PS is less than 2%, for serotype 6B the ratio ofprotein/PS is less than 4% and the ratio of NA/PS is less than 1%, forserotype 7F the ratio of protein/PS is less than 5% and the ratio ofNA/PS is less than 2%, for serotype 9V the ratio of protein/PS is lessthan 2% and the ratio of NA/PS is less than 1%, for serotype 14 theratio of protein/PS is less than 3% and the ratio of NA/PS is less than2%, for serotype 18C the ratio of protein/PS is less than 2% and theratio of NA/PS is less than 2%, for serotype 19A the ratio of protein/PSis less than 2% and the ratio of NA/PS is less than 2%, for serotype 19Fthe ratio of protein/PS is less than 3% and the ratio of NA/PS is lessthan 2%, and for serotype 23F the ratio of protein/PS is less than 2%and the ratio of NA/PS is less than 2%. Methods for the quantificationof protein, polysaccharide, and nucleic acid concentrations in acellular lysate or solution are well known in the art and include, forexample, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide GelElectrophoresis) analysis, HPLC (High Performance Liquid Chromatography)and SEC (Size Exclusion Chromatography), modified Lowry assays,spectrophotometry, SEC-MALLS (Size-Exclusion Chromatography/Multi-AngleLaser Light Scattering), and NMR (Nuclear Magnetic Resonance).

Within the methods of the present invention, the bacterial cells may belysed using any lytic agent. A “lytic agent” is any agent that aids incell wall breakdown and release of autolysin which causes cellular lysisincluding, for example, detergents. As used herein, the term “detergent”refers to any anionic or cationic detergent capable of inducing lysis ofbacterial cells. Representative examples of such detergents for usewithin the methods of the present invention include deoxycholate sodium(DOC), N-lauryl sarcosine (NLS), chenodeoxycholic acid sodium, andsaponins

In one embodiment of the present invention, the lytic agent used forlysing bacterial cells is DOC. DOC is the sodium salt of the bile aciddeoxycholic acid, which is commonly derived from biological sources suchas cows or oxen. DOC activates the LytA protein, which is an autolysinthat is involved in cell wall growth and division in Streptococcuspneumoniae. The LytA protein has choline binding domains in itsC-terminal portion, and mutations of the lytA gene are known to produceLytA mutants that are resistant to lysis with DOC.

Although there is no evidence that the use of DOC during Streptococcuspneumoniae polysaccharide purification poses a health risk, the use ofsuch biologically derived reagents could raise potential regulatoryconcerns. Accordingly, in one embodiment of the present invention, thelytic agent used for lysing bacterial cells is a non-animal derivedlytic agent. Non-animal derived lytic agents for use within the methodsof the present invention include agents from non-animal sources withmodes of action similar to that of DOC (i.e., that affect LytA functionand result in lysis of Streptococcus pneumoniae cells). Such non-animalderived lytic agents include, but are not limited to, analogs of DOC,surfactants, detergents, and structural analogs of choline, and may bedetermined using procedures as described in the Experimental sectionherein below. In one embodiment, the non-animal derived lytic agent isselected from the group consisting of decanesulfonic acid,tert-octylphenoxy poly(oxyethylene)ethanols (e.g. Igepal® CA-630, CAS #:9002-93-1, available from Sigma Aldrich, St. Louis, MO.), octylphenolethylene oxide condensates (e.g. Triton® X-100, available from SigmaAldrich, St. Louis, Mo.), N-lauryl sarcosine sodium (NLS), lauryliminodipropionate, sodium dodecyl sulfate, chenodeoxycholate,hyodeoxycholate, glycodeoxycholate, taurodeoxycholate,taurochenodeoxycholate, and cholate. In another embodiment, thenon-animal derived lytic agent is NLS.

The present invention also relates to serotype-specific modifications tothe process described above. For example, because the serotype 19Apolysaccharide is unstable and its molecular weight changes duringpurification, it was discovered that modifications to the processdescribed were useful in stabilizing the 19A polysaccharide. Thesemodifications included carrying out the ultrafiltration anddiafiltration step prior to acidification at about 4 ° C. at a pH ofabout 6 in sodium phosphate buffer, holding the acidified retentatesolution for at least 2 hours at about 4 ° C. to allow settling of theprecipitate, and adjusting the pH of the clarified polysaccharidesolution to 6 prior to the activated carbon filtration step. Bycontrast, it was discovered that for serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19F, and 23F, less polysaccharide loss and more protein removalwas achieved when the ultrafiltration and diafiltration step prior toacidification was carried out in salt free media such as water, and thisstep could be carried out at room temperature at neutral pH.

Accordingly, the present invention also relates to a process forproducing a solution containing substantially purified capsularpolysaccharides from a Streptococcus pneumoniae cell lysate comprisingserotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, or 23F comprising thesteps of:

(a) providing a fermentation broth comprising bacterial cells thatproduce Streptococcus pneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19F, or 23F;

(b) lysing the bacterial cells in step (a) with a detergent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) at room temperature at neutral pH in salt free media to remove lowmolecular weight impurities and increase polysaccharide concentration,thereby producing a salt free retentate;

(e) lowering the pH of the salt free retentate of step (d) to less than4.5, particularly about 3.5, to precipitate protein and nucleic acids,thereby forming an acidified retentate solution;

(f) holding the acidified retentate solution formed in step (e) for atleast 2 hours at room temperature with or without agitation to allowsettling of the precipitate, followed by filtration or centrifugation ofthe acidified retentate solution, thereby producing a clarifiedpolysaccharide solution;

(g) filtering the clarified polysaccharide solution of step (f) throughan activated carbon filter, particularly an activated carbon filtercomprising wood-based phosphoric acid-activated carbon;

(h) ultrafiltering and diafiltering the filtered solution produced bystep (g), thereby producing a concentrated purified polysaccharidesolution; and

(i) filtering the concentrated purified polysaccharide solution producedby step (h) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides comprising serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,19F, or 23F is produced. In a particular embodiment, step (e) removes atleast 98% of protein from the salt free retentate of step (d). Inanother embodiment, step (g) removes at least 90% of the protein fromthe clarified polysaccharide solution of step (f). In anotherembodiment, the diafiltration of step (h) comprises a pH adjustment tobetween about 5.5 to about 7.5. For improved stability of thesubstantially purified capsular polysaccharide during long-term storage,however, the diafiltration of step (h) comprises a pH adjustment tobetween about 7.0 to about 7.5, and more particularly to about 7.4.

The present invention also relates to a process for producing a solutioncontaining substantially purified capsular polysaccharides from aStreptococcus pneumoniae cell lysate comprising serotype 19A comprisingthe steps of

(a) providing a fermentation broth comprising bacterial cells thatproduce Streptococcus pneumoniae serotype 19A;

(b) lysing the bacterial cells in step (a) with a detergent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides;

(c) clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate;

(d) ultrafiltering and diafiltering the clarified cell lysate of step(c) at about 4 ° C. at a pH of about 6 in sodium phosphate buffer, 25 mMsodium phosphate, to remove low molecular weight impurities and increasepolysaccharide concentration, thereby producing a retentate;

(e) lowering the pH of the retentate of step (d) to less than 4.5,particularly about 3.5, to precipitate protein and nucleic acids,thereby forming an acidified retentate solution;

(f) holding the acidified retentate solution formed in step (e) for atleast 2 hours at about 4° C. with or without agitation to allow settlingof the precipitate, followed by filtration or centrifugation of theacidified retentate solution, thereby producing a clarifiedpolysaccharide solution;

(g) adjusting the pH of the clarified polysaccharide solution of step(f) to about 6, thereby producing a pH-adjusted clarified polysaccharidesolution;

(h) filtering the pH-adjusted clarified polysaccharide solution of step(g) through an activated carbon filter, particularly an activated carbonfilter comprising wood-based phosphoric acid-activated carbon;

(i) ultrafiltering and diafiltering the filtered solution produced bystep (h), thereby producing a concentrated purified polysaccharidesolution; and

(j) filtering the concentrated purified polysaccharide solution producedby step (i) using a sterile filter;

whereby a solution containing substantially purified capsularpolysaccharides comprising serotype 19A is produced. In a particularembodiment, step (e) removes at least 98% of protein from the retentateof step (d). In another embodiment, step (h) removes at least 90% of theprotein from the pH-adjusted clarified polysaccharide solution of step(g). In another embodiment, the diafiltration of step (i) comprises a pHadjustment to between about 5.5 to about 7.5. For improved stability ofthe substantially purified 19A polysaccharide during long-term storage,however, the diafiltration of step (i) comprises a pH adjustment tobetween about 7.0 to about 7.5, and more particularly to about 7.4.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following Examples present results for S. pneumoniae polysaccharideserotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F purified at 10L scale using the improved process of the present invention, and resultsare compared with those of the current purification process.

EXAMPLE 1 Shortened Purification Process for S. pneumoniaePolysaccharide Serotypes 1, 4, 5, 6A, 6B, 7F, 14 and 19A

S. pneumoniae fermentation broths lysed with deoxycholate sodium (DOC)were obtained either within two days following their harvest or storedin 4° C. and processed within the following week. As described below,the present purification process included the following changes comparedto the existing purification process: 1) the acidification step wasmoved from the beginning to after the firstultrafiltration/diafiltration (UF/DF) step, and the pH was adjusted to3.5 instead of 5; 2) the diafiltration buffer was changed from 0.025 Mphosphate to de-ionized (DI) water; 3) carbon adsorption was changed to2 CUNO R32SP carbon disks (CUNO Inc., Wayne, N.J.) using wood-basedphosphoric acid-activated carbon, and the adsorption time was extendedfrom 4 to 12 turnovers (each turnover was 22 minutes); and 4) pH wasadjusted to 7.4 during the last 30K diafiltration step when diafilteredabout 5 times. The same purification procedure was applied to serotypes1, 4, 5, 6A, 6B, or 7F. For serotype 19A, the steps were modifiedfurther and the purification was conducted in a chill room, as describedbelow.

Purification Steps

All steps were conducted at room temperature except for type 19A, inwhich case the process was conducted at 4° C. in a chill room.

Clarification of the lysate: The purpose of this step was to remove celldebris and clarify the broth. This was accomplished either bycentrifugation or filtration. The broth was centrifuged at 10,000 g for30 min or until the broth was clear at 20° C. (4° C. for type 19A), orfiltered with a Millipore Prefilter (Millipore Corp., Billerica, Mass.)with the addition of Celpure° filter aids (Advanced Minerals, SantaBarbara, Calif.). The clarified lysate was collected for furtherprocessing and the pellets were discarded.

First UF/DF (Ultrafiltration/Diafiltration): This step provided volumereduction and buffer exchange and also removed low molecular weightimpurities. The clarified lysate was concentrated to about ⅛^(th)original volume. Diafiltration was performed using about 10 volumes DIwater (pH 6, 25 mM phosphate for 19A).

Acidification: More than 98% of the proteins were removed in this step.While stirring, concentrated phosphoric acid was added carefully to theretentate. The pH of the retentate was adjusted to a target value of pH3.5. The acidified retentate was stirred for half an hour and aged inroom temperature to age overnight (2 hours at 4° C. for 19A), resultingin the precipitation of protein and nucleic acids.

Clarification of Acidified Retentate: This was the clarification step toremove the precipitates after acidification. The slurry of acidifiedsolution was centrifuged in a rotor at 10,000 rpm (17,000 RelativeCentrifugal Force or RCF) for one hour at 20° C. (except for 6B, inwhich case the centrifugation was 6 hours at 37° C.). The supernatantwas collected and the pellet was discarded. Depth filtration with aMillipore Prefilter (Millipore Corp., Billerica, Mass.) with theaddition of Celpure® filter aids (Advanced Minerals, Santa Barbara,Calif.) can also be used for this step.

Carbon Adsorption: In most cases, there was a slight yellow color aftercentrifugation of the acidified 100K retentate. The color removal wasachieved by the carbon adsorption using wood-based phosphoricacid-activated carbon. This step also removed residual protein thatremained after acidification. The clarified polysaccharide solution wasrecirculated through the carbon filter for 5-6 hours or overnight (for19A, the pH was adjusted to 6 before carbon adsorption).

Final 30K UF/DF: This was another concentration and buffer exchange stepto concentrate the solution to a final polysaccharide (PS) concentrationof >2 g/L, which was diafiltered into deionized (DI) water. Thecarbon-filtered PS solution was concentrated. Then the concentrate wasdialfiltered 10× with DI water. The pH was adjusted to 7.4 during thediafiltration.

Final 0.2 μ m Sterile Filtration: The final PS solution was sterilefiltered with a 0.22 μ m filter or sterile disposable filter unit andstored in a 4° C. refrigerator.

Analytical Methods

Quantification of protein, PS, and nucleic acid concentrations werecarried out using methods that are well known in the art, includingSDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)analysis, HPLC (High Performance Liquid Chromatography) and SEC (SizeExclusion Chromatography), modified Lowry assays, spectophotometry,SEC-MALLS (Size-Exclusion Chromatography/Multi-Angle Laser LightScattering), and NMR (Nuclear Magnetic Resonance).

Results and Discussion

Type 5 Shortened Purification Batches: The comparison of final PS yield,PS molecular weight and major impurity levels of three purificationbatches using the shortened process and those using the current processfor Type 5 are shown in Table 1. All the starting broths were DOC lysedhigh cell density fermentation batches, which had a higherpolysaccharide concentration (˜0.5 g/L) than the standard SOPfermentation broth (˜0.3 g/L). A 30K or 50K membrane was used for thefirst UF/DF step. The impurity levels were calculated using impurity/PSratios. The same approach was used for all other serotypes describedherein.

TABLE 1 Testing Data for Type 5 Shortened Purification Process Batches.PS PS PROTEIN NA MOLECULAR C-PS YIELD RATIO RATIO WEIGHT RATIO BATCH #(%) (%) (%) (KG/MOL) (%) L29276-27 56.7 1.2 0.12 299 20.7 (30K UF/DF)L29276-32 54.6 1.8 0.12 282 24.7 (30K UF/DF) L29276-52 60.2 1.4 0.03 27522.8 (50K UF/DF) Current process 57 3.6 0.18 320 15.9 Specification or50-60 ≦7.5 ≦2.0 NA ≦35 expected value

As the data in Table 1 show, the protein ratios of all three batchesusing the shortened purification process met the specification of ≦7.5%,and were also comparable to that of the current process. The nucleicacid (NA) as well as C-polysaccharide (C-PS) ratios were also well belowthe specifications of ≦2.0% and ≦35%, respectively and were comparableto that of the current process. The results of the final PS yield andimpurity levels of the three batches shown in Table 1 demonstrate thereproducibility and robustness of the shortened process.

There was some concern about whether polysaccharides could be hydrolyzedat a lower pH of 3.5. PS retention time change during the purificationprocess was therefore monitored, and the molecular weight of the finalpurified PS was measured. No significant changes in serotype 5 PSretention time from the HPLC chromatogram were observed. There were alsono significant differences in molecular weights for the purified PS bythe shortened process and that of the current process (284 kg/mol) basedon the MALLS measurement. Thus it was concluded that the molecularweight of purified PS was not adversely affected by the shortenedpurification process.

TABLE 2 Type 5 In-Process PS yield, Protein (SDS-PAGE) and Nucleic AcidRatios. PS YIELD (%) PROTEIN/PS (%) STEP L29276-27 L29276-32 L29276-52L29276-27 L29276-32 L29276-52 Broth 100.0 100.0 100.0 384.40 1026.60193.26 Centrifugation 99.9 95.6 94.6 274.60 642.00 209.78 30/50K UF/DF71.1 82.8 93.9 152.10 561.00 204.76 Acidification 56.1 65.9 58.6 9.007.50 0.20 Carbon 57.7 55.7 58.7 0.20 1.90 0.50 30K UF/DF 54.5 54.6 60.30.10 1.00 0.09 NUCLEIC ACID/PS (%) STEP L29276-27 L29276-32 L29276-52Broth 825.80 579.83 344.70 Centrifugation 505.10 484.30 311.90 30/50KUF/DF 107.30 139.90 137.60 Acidification 9.44 16.40 7.38 Carbon 0.220.30 0.74 30K UF/DF 0.11 0.12 0.19

The in-process polysaccharide yield, protein/polysaccharide ratio andnucleic acid/polysaccharide ratio at each of the processing steps forthe three shortened process batches are summarized in Table 2.

There is always a loss of product during each step of any purificationprocess. For these three shortened process batches, PS loss occurredmostly in the first UF/DF step and the acidification step. The loss ofPS at the first UF/DF step was due to adsorption of PS or PS-proteincomplex to the surface of the membrane. This loss was minimized byrinsing the retentate side of the membrane with DI water after thediafiltration and combining the rinse with the original retentate. Theloss of PS at the acidification step could be due to two reasons:physical adsorption of PS to the precipitation solids, andpolysaccharide binding to the protein with co-precipitation duringacidification. This second possibility was further investigated andresults showed evidence of PS/protein binding.

FIG. 1 shows the reduction of protein/PS ratio at each purificationstep. Although the centrifugation step removed a small percentage ofproteins, the majority of protein was removed at the acidification step.Only a trace amount of protein was detected after pH 3.5 treatment evenfor the highest protein potency batch.

Similar to protein/PS ratio, the nucleic acid/PS ratio showedvariability of impurity levels among batches. The 30/50K UF/DF stepremoved a significant amount of nucleic acid as compared to proteinremoval in the same processing step. Without being bound by theory, thismay have been due to the molecular size of nucleic acids being smallerthan that of proteins, making them relatively easier to be removed viathe 30/50K UF/DF step. The first centrifugation and acidification stepalso removed a considerable amount of nucleic acid.

Table 2 shows that the activated carbon adsorption step also reducesprotein/PS and NA/PS level. The percentage reduction was not assignificant as the first two steps, but this step was important forremoving the color of the solution and ensured that the impurity levelmet the specification. Type 4 Shortened Purification Batches: A summaryof three shortened purification batches for Type 4 is shown in Table 3.The feed broths for all three batches were DOC-lysed.

TABLE 3 Type 4 Shortened Purification Batches Summary. PS PS PRO-NUCLEIC MOLECULAR C-PS YIELD TEIN/PS ACID/PS WEIGHT RATIO BATCH (%) (%)(%) (KG/MOL) (%) L29276-47 56.3 0.7 0.04 289 15.4 L29276-55 53.0 1.30.03 354 14.7 L29276-148 57.3 1.16 0.02 293 10.0 Current 71.1 0.05 0.02285 process Specification NA <3.0 <2.0 >350 <25

The type 4 PS yield was also between 50-60%. Protein/PS ratio andnucleic acid/PS ratio were well within their specifications. C-PS ratiowas also well within the specification. The molecular weight of all ofthe three batches were close to ˜300 kg/mol. Serotype 4 PS purified bythe current purification process using the similar fermentation brothalso gave a lower molecular weight 285 kg/mol. Comparison of the HPLCchromatographs of the PS from fermentation broth and the final purifiedsolution showed that there were no differences in the PS retention time.This suggested that the difference in molecular weight difference wasnot caused by the process change, but rather due to intrinsic nature ofthe fermentation process.

The Type 4 PS contains a pyruvate group in the purified molecule, andthis pyruvate was important for conjugation for use in a pneumococcalconjugate vaccine. To ensure that the pyruvate amount was not adverselyaffected by acid treatment, NMR analysis was conducted. FIG. 2A and FIG.2B shows the NMR spectra for standard type 4 PS and that from batchL29276-47. No significant differences between the two spectra wereobserved. The second peak from the right in both spectra was pyruvate,and the pyruvate group peak height was comparable in both spectra. Thepyruvate ratios for all three shortened process batches were 0.8mol/mol, and met the specification of >0.7 mol/mol.

The in-process PS yield, protein/PS and NA/PS ratio for the three type 4batches are summarized in Table 4. PS loss occurred mostly at the firstcentrifugation, acidification and activated carbon adsorption steps,with an average of 10%, 8% and 20%, respectively. The overall PS yieldwas close to that of type 5, around 55%.

TABLE 4 Type 4 In-Process PS Yield, Protein (SDS-PAGE)/PS Ratio, andNA/PS Ratio. PS YIELD (%) PROTEIN/PS (%) STEP L29276-47 L29276-55L29276-148 L29276-47 L29276-55 L29276-148 Broth 100.0 100.0 100.0 231.22297.33 499.75 Centrifugation 85.8 97.8 88.0 198.69 247.40 534.61 50/100KUF/DF 85.1 86.4 87.7 235.90 249.24 364.98 Acidification 84.2 72.0 78.60.42 1.81 0.41 Carbon 65.3 48.9 58.6 0.09 0.39 0.34 30K UF/DF 57.9 50.657.4 0.08 0.09 1.31 NA/PS (%) STEP L29276-47 L29276-55 L29276-148 Broth312.84 282.50 302.71 Centrifugation 301.40 261.82 243.28 50/100K UF/DF80.99 67.67 92.00 Acidification 4.24 5.01 1.17 Carbon 0.08 0.92 0.38 30KUF/DF 0.05 0.06 0.07

FIG. 3 shows average PS yield, protein/PS and NA/PS ratio change at eachof the purification steps for the three batches in Table 4. The proteinremoval occurred mostly at the acidification step as expected. The firstcentrifugation and UF/DF steps also collectively removed a certainamount of protein, but the protein reduction was less than theacidification step.

Similar to type 5, the most nucleic acid/PS ratio reduction took placeat the 50/100K UF/DF, first centrifugation, and the acidification steps,and the NA reduction at the first UF/DF step was more significant thanthat for proteins. Again, as shown in FIG. 3, the activated carbon stepremoved a certain amount of protein and NA and brought the impuritylevels below the specifications. It also removed the color of thesolution as well.

Type 19A Shortened Purification Batches: Type 19A polysaccharide isunstable and the molecular weight changes during purification. Theshortened purification process was modified slightly in order tostabilize the 19A polysaccharide. These modifications are summarized asfollows: 1) the purification steps were mostly conducted at 4° C. in thechill room; 2) the first 100K diafiltration was chilled using 25 mMphosphate buffer (4° C.) with a pH of 6 instead of using roomtemperature water; 3) the acidification holding time was reduced fromovernight to 2 hours; and 4) after clarifying the acidified 100Kretentate, the pH was adjusted to 6 and activated carbon adsorption wasconducted at pH 6 instead of 3.5.

The results of two 19A batches purified by the shortened purificationprocess are shown in Table 5.

TABLE 5 Type 19A Shortened Purification Batches Summary. PS MOLECULARYIELD PROTEIN/PS NA/PS WEIGHT C-PS BATCH (%) (%) (%) (KG/MOL) (%)L29276-116 61.58 0.14 0.01 525 3.8 L29276-143 76.15 1.43 0.01 488 2.8Specification NA <2 <2 NA <10

The PS yields of the two shortened purification batches were 62 and 76%respectively. Final protein/PS ratio, nucleic acid ratio, and C-PS ratioall met their respective specifications. The final molecular weights ofpolysaccharides from the two batches were 525 kg/mol and 488 kg/mol,respectively, and were close to that of the PS of 19A used in phase IIIclinical trials (486kg/mol).

The protein/PS ratio for the two batches both met the specification of<2%. Both NA and C-PS ratio of the two batches were well within theirspecifications.

The PS yield, protein and NA reduction at each of the purification stepsare shown in Table 6 and FIG. 4. The results showed PS loss at each ofthe purification steps except the first centrifugation step. Likeserotypes 5 and 4, protein and NA removal mostly took place at the firstthree steps, and there was hardly any detectable protein and NA leftafter acidification. Although the activated carbon adsorption step didnot remove a significant amount of protein and NA, possibly due to verylow protein and NA concentration after the acidification, the step wasstill needed for color removal.

TABLE 6 Type 19A In-Process PS Yield, Protein/PS and NA/PS Ratio. PSYIELD (%) PROTEIN/PS (%) NA/PS (%) STEP L29276-116 L29276-143 L29276-116L29276-143 L29276-116 L29276-143 Broth 100.0 100.0 224.41 144.08 277.55134.73 Centrate 100.8 101.2 121.60 119.11 189.64 100.64 100K UF/DF 87.790.3 103.83 105.16 56.87 26.36 Acidification 90.4 72.1 0.28 0.84 0.030.03 Carbon 77.8 71.7 0.96 0.03 0.04 0.02 30K UF/DF 61.0 76.1 0.54 0.000.02 0.01

Type 7F Shortened Purification Batches: Type 7F is a non-ionicpolysaccharide, which typically requires change in steps duringpurification using the existing process compared to the serotypesdescribed above). However, the shortened purification process of thepresent invention was successfully applied to serotype 7F without theneed for process deviation. Two batches of type 7F broth were purifiedusing the shortened process. One was a standard fermentation broth(L29276-107) and one was a high cell density broth (L29276-157). Theresults of the two batches are summarized in Table 7.

TABLE 7 Type 7F Shortened Purification Batches Summary. PS MOLECULARYIELD PROTEIN/PS NA/PS WEIGHT C-PS BATCH # (%) (%) (%) (KG/MOL) (%)L29276-107 65.27 0.49 0.02 968 3.3 L29276-157 79.06 0.26 0.04 881 2.9Specification NA <5 <2 NA <17

The PS yield of type 7F was actually higher than the other serotypes,possibly due to less binding of the non-ionic polymer to the chargedprotein molecules. Final protein, NA and C-PS ratios were all wellwithin their specifications. Molecular weight of type 7F was comparableto that of standard batches and even though the 7F molecular weight wasquite high, the PS solution was not very viscous due to smaller excludedvolume of the nonionic polymer.

Type 7F PS yield, protein and NA ratio at each of the purification stepsare shown in Table 8 and the average of the two batches was plotted inFIG. 5.

TABLE 8 Type 7F In-Process PS Yield, Protein/PS, and NA/PS Ratio. PSYIELD (%) PROTEIN/PS (%) NUCLEIC ACID/PS (%) STEP L29276-107 L29276-157L29276-107 L29276-157 L29276-107 L29276-157 Broth 100.00 100.00 274.10189.24 258.55 134.64 Centrifugation 100.31 95.57 110.60 107.20 163.3883.14 100K UF/DF 84.19 95.39 139.38 27.53 54.02 32.74 Acidification70.89 84.08 0.44 0.10 1.80 0.94 Carbon 63.96 79.40 0.70 0.09 2.29 0.2330K UF/DF 57.24 77.70 0.30 0.05 0.06 0.00

There was some Type 7F PS loss at each purification step. Overall, PSloss was less than that of serotypes 5 and 4. Protein and NA ratioreduction was mostly by the first centrifugation, 100K UF/DF, andacidification steps. Similar to other serotypes, the 100K UF/DF was moreefficient in removing NA than proteins. Although the activated carbonadsorption step did not remove a significant amount of protein and NAdue to very low impurity levels after acidification, the step was stillneeded for color removal.

Type 6B Shortened Purification Batches: Two batches of 6B were purifiedusing the shortened purification process. It was found the clarificationof the acidified 100K retentate took a longer time than the otherserotypes (6 hours instead of 1 hour). Except for this difference, thepurification process was similar to that of the serotypes. The resultsof the two batches are summarized in Table 9.

TABLE 9 Type 6B Shortened Purification Summary. PS MOLECULAR YIELDPROTEIN/PS NA/PS WEIGHT C-PS BATCH (%) (%) (%) (KG/MOL) (%) L29276-16169.50 1.73 0.28 857 3.6 L29276-164 74.50 1.41 0.17 1142 3.9Specification NA <4 <1 >800 <10

All impurity levels (protein, NA, and C-PS) were well within theirspecifications. The PS yield was relatively high, and so were theprotein and NA ratio compared to the other serotypes.

The in-process PS yield, protein and NA ratios at each of thepurification steps are shown in Table 10 and FIG. 6.

TABLE 10 Type 6B Average In-Process PS Yield, Protein, and NA Ratio. PSYIELD (%) PROTEIN/PS (%) NUCLEIC ACID/PS (%) STEP L29276-161 L29276-164L29276-161 L29276-164 L29276-161 L29276-164 Broth 100.00 100.00 238.20288.80 215.12 237.67 Centrifugation 101.43 116.54 95.88 123.59 104.08104.47 100K UF/DF 104.11 105.47 68.15 88.14 22.13 47.81 Acidification91.98 101.45 0.71 0.43 1.06 2.24 Carbon 80.71 91.42 0.39 0.23 0.45 0.7430K UF/DF 72.67 77.28 1.23 0.46 0.37 0.25

Similar to 19A, PS loss occurred at each of the purification stepsexcept the first centrifugation step, in which there was a slightincrease of PS. Removal of protein and NA was achieved mostly by thefirst centrifugation, the first 100K UF/DF, and acidification steps.However, there was also some reduction of protein and NA ratio at theactivated carbon adsorption step.

Type 6A Shortened Purification Batches: Two batches of type 6A werepurified using the shortened purification process. The PS yield,impurity levels and molecular weight of the final solutions aresummarized in Table 11.

TABLE 11 Type 6A Shortened Purification Batches Summary. MOLECULAR PSYIELD PROTEIN/PS NA/PS WEIGHT C-PS BATCH BROTH (%) (%) (%) (KG/MOL) (%)L29276-138 Standard 75.75 1.23 0.04 640 7.3 L29276-183 High cell density72.56 0.20 0.01 670 5.1 Specification NA <2.00 <2.00 NA <15

The final PS yields of the two 6A batches were both >70%, which were thehighest among the serotypes processed using the shortened process.Protein, NA, and C-PS ratios were all within specifications.

Table 12 and FIG. 7 show in-process PS yield, protein and NA ratiochange at each of the purification steps. There was hardly any loss ofPS at the first centrifugation and 100K UF/DF steps. About 10-15% PSloss occurred at the acidification and activated carbon adsorptionsteps, which was close to that of the other serotypes. The most proteinand NA ratio reduction was by the first centrifugation, 100K UF/DF, andacidification. After acidification, both protein and NA ratios werealready below their specifications. For protein, the acidification wasthe most efficient step, while for NA, 100K UF/DF reduced the most NA/PSratio. Although the activated carbon adsorption step did not remove asignificant amount of protein and NA due to very low impurity levelsafter acidification, the step was still needed for color removal.

TABLE 12 Type 6A In-Process PS Yield, Protein and NA Ratio. PS YIELD (%)PROTEIN/PS (%) NUCLEIC ACID/PS (%) STEP L29276-138 L29276-183 L29276-138L29276-183 L29276-138 L29276-183 Broth 100.00 100.00 214.35 138.71211.45 163.88 Centrifugation 99.35 97.80 183.43 149.71 177.03 150.41100K UF/DF 93.19 100.40 114.72 103.00 43.38 40.43 Acidification 89.8787.90 0.16 0.87 1.09 0.97 Carbon 76.02 75.70 0.62 0.85 0.09 0.07 30KUF/DF 75.57 72.90 0.00 0.07 0.04 0.02

Type 1 Shortened Purification Batches: Two batches of type 1 purified bythe shortened process are summarized in Table 13. Batch L29276-170 waspurified from high cell density fermentation broth and L29276-173 wasfrom standard fermentation broth.

TABLE 13 Type 1 Shortened Purification Batch Summary. MOLECULAR PS YIELDPROTEIN/PS NA/PS WEIGHT C-PS BATCH BROTH (%) (%) (%) (KG/MOL) (%)L29276-170 High cell density 50.96 0.44 0.08 501 5.3 L29276-173 SOP53.84 1.23 0.01 458 11.9 Specification NA <2 <2 NA <15

The PS yields of both batches were about 50%, and the impurity levelsfor protein, NA and C-PS were all within their specifications.

The in-process PS yield, protein and NA ratios after each of thepurification steps are shown in Table 14 and FIG. 8. The trends weresimilar to other serotypes purified. PS loss occurred mostly at theacidification and activated carbon adsorption steps, and most of proteinand NA removal occurred at the first three steps. More NA was removed atthe 100K UF/DF step than proteins. Although the activated carbonadsorption step did not remove a significant amount of protein and NAdue to very low impurity levels after acidification, the step was stillneeded for color removal.

TABLE 14 Type 1 Average In-Process PS Yield, Protein and NA Ratio. PSYIELD (%) PROTEIN/PS (%) NA/PS (%) STEP L29276-170 L29276-173 L29276-170L29276-173 L29276-170 L29276-173 Broth 100.00 100.00 453.30 595.20594.41 1178.98 Centrifugation 93.99 105.63 301.86 396.97 340.55 1018.4750/100K UF/DF 88.33 105.62 232.34 235.08 17.49 182.56 Acidification66.54 87.67 0.54 4.87 1.09 5.85 Carbon 55.85 59.69 1.91 1.86 0.49 0.0130K UF/DF 51.24 55.59 0.36 0.92 1.65 0.01

Type 14 Shortened Purification Batches: Two batches of serotype 14 werepurified using the shortened purification process with no processdeviations. The final PS yield, and impurity levels are summarized inTable 15.

TABLE 15 Type 14 Shortened Purification Batches Summary. MOLECULAR PSYIELD PROTEIN/PS NA/PS WEIGHT C-PS BATCH BROTH (%) (%) (%) (KG/MOL) (%)L32874-155 High cell density 51.3 2.06 0.03 520 4.2 L32874-163 SOP 56.71.39 0.03 662 2.9 Specification NA <3 <2 >400 <15

Similar to serotype 7F, serotype 14 is a non-ionic polysaccharide, andits current purification process is slightly different from the otherserotypes. However, the shortened purification process of the presentinvention was successfully applied to serotype 14 without the need forsuch additional steps. The PS yields of the two shortened purificationprocess batches were 50-60%, and protein and NA ratios were within theirspecifications. The molecular weights of the purified PS also met thespecification.

In-process PS yield, protein and NA ratios are summarized in Table 16and FIG. 9. Similar trends of PS loss, protein and NA removal wereobserved as for the other tested serotypes described above.

TABLE 16 Type 14 Average In-Process PS Yield, Protein, and NA Ratio. PSYIELD (%) PROTEIN/PS (%) L32874- L232874- L32874- L232874- STEP 155 163155 163 Broth 100.00 100.00 109.73 286.56 Centrifugation 91.40 90.80110.88 201.48 50/100K UF/DF 82.40 68.70 45.52 165.69 Acidification 65.4093.90 0.65 0.47 Carbon 59.40 74.60 0.60 0.10 30K UF/DF 53.10 57.60 0.570.41 NA/PS (%) L32874- L232874- STEP 155 163 Broth 192.80 272.50Centrifugation 135.50 201.82 50/100K UF/DF 47.10 64.54 Acidification0.89 1.08 Carbon 0.17 0.21 30K UF/DF 0.13 0.25

EXAMPLE 2 Comparison of Different Serotypes (Serotypes 1, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, and 19F)

To compare purification of the different serotypes using the shortenedpurification process of the present invention, PS removal for all theserotypes described in Example 1 plus serotypes 9V, 18C, and 19F wereplotted in one graph in FIG. 10. Most of the serotypes followed asimilar trend with PS loss at each of the purification steps. There wasan increase of PS yield for type 6B at the first centrifugation step andtype 14 at the acidification step. PS percentage loss varied fromserotype to serotype. Type 5 seemed to lose most PS at the acidificationstep and type 4 at the activated carbon adsorption step.

Protein/PS ratios for each purification step for each of the serotypesare shown in FIG. 11. The figure shows that there was a difference ininitial protein/PS ratios for different serotypes, with types 1, 4, 5,9V, 19F, and 18C having the highest initial protein/PS ratios. Eventhough the protein/PS ratios were much higher for types 1, 4, 5, 9V,19F, and 18C compared to the other serotypes even after the first UF/DFstep, the acidification step greatly reduced the protein/PS ratio andnot much protein was left after this step.

As shown in FIG. 12, NA/PS ratios also varied from serotype to serotype.Types 1 and 5 had the highest NA/PS ratio, followed by types 18C and 4.The first centrifugation and UF/DF step removed a significant amount ofNA for these serotypes and type 1 seemed to be the most efficient. Therewas very little NA left after the acidification step.

EXAMPLE 3 Acidification and Activated Carbon Adsorption Step EfficiencyAnalysis (Serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F)

For purification of PnC polysaccharides, the most significant anddifficult impurity to remove is proteins. To better understand theimpurity removal efficiency of the shortened process, two of the majorpurification steps, acidification and activated carbon adsorption, wereanalyzed for protein removal. For the acidification step, the differencein protein concentration (SDS-PAGE) before and after acidification wasplotted against the initial protein concentration before acidificationfor serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F using theshortened purification process of the invention (FIG. 13).

Due to relatively small changes in solution volume before and after theacidification step, the protein concentration difference divided by theinitial protein concentration reflected the protein removal rate by theacidification step. FIG. 13 shows the slope of the linear fit of proteinconcentration difference with respect to initial protein concentration(by SDS-PAGE assay). A very good linear relationship was observedbetween the protein concentration change and the initial concentration,with the R² close to 1. The slope was 0.9876, which corresponds to a98.76% protein removal (assuming negligible solution volume change).Therefore, for all serotypes studied, the acidification step was veryefficient and on average it removed more than 98% protein.

Similar to the acidification step, the efficiency of the activatedcarbon adsorption step was also evaluated by plotting the amount ofprotein removed (adsorbed on carbon) with respect to initial proteinloading (FIG. 14). A good linear relationship between the removedprotein and the initial protein loading was observed, although the R²(0.9723) was not as high as for the acidification step. Because theslope of this linear fit corresponds to the protein removal rate, theactivated carbon adsorption step produced 93.87% protein removal.

Based on an analysis of the acidification and activated carbonadsorption steps, after the two steps only about 0.1% protein was leftin the solution.

CONCLUSIONS FOR EXAMPLES 1-3

A shortened purification process was developed to replace the currentpurification process for capsular polysaccharides of S. pneumoniae. Theshortened purification process was directly applied to serotypes 1, 4,5, 6A, 6B, 7F, 9V, 14, 18C, and 19F, and to 19A with slight deviation.The process was conducted at 10 L scale using DOC-lysed fermentationbroths. The impurity levels, including protein/PS, NA/PS and C-PS/PSratios, all met their respective specifications. The PS yields were over50% and comparable to that of the current purification process.In-process PS yield, protein/PS and NA/PS ratios were plotted to comparethe behavior of different serotypes. Serotypes 1, 5, 9V, 19F, and 18Cwere found to be the most difficult to purify based on protein/PS ratiosbefore and after the 100K UF/DF step.

Step analysis showed that the acidification and activated carbonadsorption steps removed more than 98% and 90% protein, respectively (asmeasured by SDS-PAGE).

EXAMPLE 4 Substitution of Non-Animal Derived Lytic Agents forDeoxycholate Sodium in the Production of Polysaccharides

The present example investigated whether non-animal derived lytic agentscould be used as a substitute for deoxycholate sodium (DOC) within theprocess described above for the production of substantially purifiedcapsular Streptococcus pneumoniae polysaccharides. As described above,DOC activates the LytA protein, which is an autolysin that is involvedin cell wall growth and division in Streptococcus pneumoniae. The LytAprotein has choline binding domains in its C-terminal portion, andmutations of the lytA gene are known to produce LytA mutants that areresistant to lysis with DOC.

A rapid microtiter plate assay was developed to identify compounds thatcause cell lysis by a mechanism similar to that of DOC. Severalnon-animal derived alternatives to DOC were identified which wereequally effective as DOC at killing Streptococcus pneumoniae cells andreleasing polysaccharide. Following processing using standardconditions, the size and purity of the polysaccharides produced with thenon-animal derived compounds were identical to those produced with DOC.

Methods

Cell Lysis: Compounds to be tested were added to fermentation broth at afinal concentration of 0.1 to 0.01% (v/v) and the solution was allowedto incubate at 37° C. for one hour. The solution was then stored at 2-8°C. overnight. The following morning the solution was centrifuged toremove all cell debris. The cell-free broth was analyzed by SEC-HPLC todetermine the concentration of released polysaccharide. The lysis couldbe performed at any temperature between 2-37° C., preferably at a pHrange of 6.0-7.5. The concentration of the detergent was typicallybetween 0.05-0.2% depending on the particular detergent.

Mitrotiter Assay: A rapid microtiter plate assay was devised forexamining lytA-dependent lysis of pneumococci by different detergents orsurfactants. Two pairs of isogenic strains of S. pneumoniae were used inthe assay; one member of each pair was wild type for the lytA gene whilethe other strain carried a deletion in the lytA gene Thus, if lysis weredependent on an active lytA function, that detergent would not lyse themutant strains. The four strains, R6X, R6X ΔlytA, D39 and D39 ΔlytA,were cultivated in HySoy medium to approximately mid-log phase(OD₆₀₀˜0.2-0.8; OD₆₀₀=Optical Density at 600 nm). Cells were thencentrifuged and cell pellets were resuspended in HySoy medium. To eachwell of the microtiter plate, 100 μL, of cell suspension was added,along with 10 μL, of detergent stocks or water as a control. After about15 minutes at 36° C., the OD₆₀₀ of the samples was measured with aSpectramax® spectrophotometer (Molecular Devices, Sunnyvale, Calif.).The following results were observed for freshly prepared cells or frozenand thawed cells: Wild-type cells exposed to DOC, sodium dodecylsulfate, Triton® X-100 and N-laurylsarcosine all had OD valuescomparable to the medium blank, which indicated that lysis had occurred.On the other hand, the ΔlytA cells did not lyse in the presence of thesedetergents, which indicates that LytA function is required for thesedetergents to lyse the cells.

Isolation of PS Used For Comparative Analytical Studies: Cultures weregrown in Hy-Soy medium in 10 L bioreactors. The pH was controlled ataround 7.0 using either NaOH or Na₂CO₃. At the end of growth (asindicated by no further increase in optical density), the cultures weretreated with either 0.12% DOC or 0.1% NLS. The cultures were incubatedfor 12-16 hours. The effectiveness of cell killing was confirmed byplating a sample of treated broth on TSA-Blood Agar plates.Polysaccharide was purified from the clarified lysate using standardprocedures (as described above). Purified polysaccharide was examinedusing a variety of standard analytical techniques appropriate todetermine the purity and identity of the material.

The PS content of the lysate was determined using SEC-HPLC coupled to arefractive index (RI) detector.

Screening for Non-Animal Derived Lytic Agents Using Serotypes 1 and 6B

S. pneumoniae serotypes 1 and 6B were separately grown in Hy-Soy basedmedia. The cultures were separately harvested and separately dispensedinto tubes. The non-animal derived compounds to be screened for lyticactivity comparable to DOC were prepared as stock solutions (in suitablesolvents) and added to the cultures. After overnight incubation, thetubes were centrifuged and the PS content of the lysates for eachserotype were determined by SEC-HPLC and compared to DOC.

Screening for Non-Animal Derived Lytic Agents Using lytA Mutants

Isogenic pairs of strains containing the lytA mutation were grown in aHy-Soy based medium. The cells were harvested and dispensed into wellsin a microttiter plate. The test compound was added and the culture wasincubated. After 15 min at 36° C., the optical density (OD₆₀₀) of eachwell was determined using a SpectraMax® plate reader (Molecular Devices,Sunnyvale, CALIF.)(see Tables 17 and 18, which summarize results fromtwo separate tests for exemplary compounds).

TABLE 17 Change in Optical Density for lytA Mutant Strains (Test 1)Change in OD₆₀₀ R6X D39 Compound R6X ΔlytA D39 ΔlytA Blank No addnDeoxycholic acid, 0.1% 100%  34% 99% 47% Lithocholic acid, 0.1% 20% 16% 8% −2% Tauroglycocholic acid, 0.1% 51% −16%  47% −20%  Heptanoic acid,0.1% 25% −57%   7% −30%  SDS, 0.1% 99% 28% 95% 36% Octanesulfonic acid,0.1% 43%  6% 34% 16% Triton ® X-100, 0.1% 91% 22% 97% 30% Tween 80, 0.1%36% 14% 20% 19% Tween 20, 0.1% 44% 22% 28% 19% N-lauryl sarcosine, 0.1%100%  −34%  89% −44%  Pluronic L31, 0.1% 39%  0% 17% −38%  PluronicL-61, 0.1% 23% −3% −19%  14% Pluronic L81, 0.1% 21% −3% −14%  10%Antarox 17-R-2, 0.1% 37% 24%  7% 16%

TABLE 18 Change in Optical Density for lytA Mutant Strains (Test 2)Change in OD₆₀₀ R6X D39 Compound R6X ΔlytA D39 ΔlytA Blank No addn Water −8%  −8%  3% −8% Deoxycholic acid, 0.1% 103%  20% 101% 41% SDS, 0.1%102% −13% 100% 16% N-lauryl sarcosine, 0.1% 102% −40% 101% 14% Triton ®X-100, 0.1% 101% −19% 100%  4% Tween 20, 0.1%  6% −17%  −5% −3%Octanesulfonic acid, 0.1%  13%  3%  18% −3%

Based on the screening studies described above, the following non-animalderived lytic agent alternatives to DOC were identified: decanesulfonicacid, Igepal® CA-630 (tert-Octylphenoxy poly(oxyethylene)ethanol; CAS #:9002-93-1; available from Sigma Aldrich, St. Louis, Mo.), N-laurylsarcosine sodium (NLS), lauryl iminodipropionate, sodium dodecylsulfate, Triton® X-100, chenodeoxycholate, hyodeoxycholate,glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate, andcholate.

Comparison of DOC-Lysed PS to NLS-Lysed PS

S. pneumoniae polysaccharide serotypes 1, 4, 5, 6A, 6B, and 7F werepurified at the 10 L scale as described above in Examples 1 and 2 usingthe improved process of the present invention. However, in one group NLS(0.1%) was used as the lytic agent while in another group DOC (0.12%)was used as the lytic agent.

PS yield, protein/PS ratios, NA/PS ratios, and PS molecular weight weremeasured as described above, and results are summarized in Table 19.These results showed that the use of NLS as a lytic agent within thepurification methods of the invention produced relatively high PS yieldswith relatively low protein and nucleic acid levels. In fact, for themajority of serotypes tested, use of NLS as a lytic agent producedhigher PS yields compared to the use of DOC.

TABLE 19 PS Characterization For Different Serotypes Using DOC vs. NLSPS Nucleic SEC MALLS Yield Protein/PS Acid/PS MW (g/mol, Serotype LyticAgent (%) (%) (%) 10⁶) 1 0.12% DOC 20% 4.2% 0.04% 0.615 1 0.1% NLS 41% 2% 0.04% 0.65 4 0.12% DOC 72% 0.05%  0.02% 0.38 4 0.1% NLS 57% 0.67% 0.25% 0.34 5 0.12% DOC 57% 3.6% 0.18% 0.32 5 0.1% NLS 63% 1.8% 0.01%0.35 6A 0.12% DOC 56% 0.7% 0.00% 0.55 6A 0.1% NLS 66% 1.1% 0.05% 0.43 6B0.12% DOC 38% 0.6% 0.05% 0.969 6B 0.1% NLS 55% 0.1% 0.01% 1.0 7F 0.12%DOC 73% 0.7% 0.06% 0.814 7F 0.1% NLS 76% 0.5% 0.02% 0.912

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A solution containing substantially purified Streptococcus pneumoniaecapsular polysaccharide wherein the percent ratio of protein topolysaccharide (protein/PS) is less than 10%.
 2. A solution according toclaim 1 wherein the percent ratio of protein/PS is less than 2%.
 3. Asolution containing substantially purified Streptococcus pneumoniaecapsular polysaccharide wherein the percent ratio of nucleic acid topolysaccharide (NA/PS) is less than 5%.
 4. A solution according to claim3 wherein the percent ratio NA/PS is less than 2%.
 5. A solutionaccording to claim 1 wherein the percent ratio of NA/PS is less than 5%.6. A solution according to claim 1 wherein the percent ratio of NA/PS isless than 2%.
 7. A solution according to claim 1 or 3 wherein thepolysaccharide is serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or23F Streptococcus pneumoniae capsular polysaccharide.
 8. A solutionaccording to claim 7, wherein the polysaccharide is serotype 1, andwherein the percent ratio of protein/PS is less than 2% and the percentratio of NA/PS is less than 2%.
 9. A solution according to claim 7,wherein the polysaccharide is serotype 4, and wherein the percent ratioof protein/PS is less than 3% and the percent ratio of NA/PS is lessthan 2%.
 10. A solution according to claim 7, wherein the polysaccharideis serotype 5, and wherein the percent ratio of protein/PS is less than7.5% and the percent ratio of NA/PS is less than 2%.
 11. A Streptococcuspneumoniae serotype 6A capsular polysaccharide, wherein said capsularpolysaccharide has a molecular weight of 640,000 daltons to 670,000daltons.
 12. A solution containing substantially purified Streptococcuspneumoniaeserotype 6A capsular polysaccharides according to claim 11.13. A solution according to claim 7, wherein the polysaccharide isserotype 6A, and wherein the percent ratio of protein/PS is less than 2%and the percent ratio of NA/PS is less than 2%.
 14. A solution accordingto claim 13, wherein said polysaccharide has a molecular weight of640,000 daltons to 670,000 daltons.
 15. A solution according to claim 7,wherein the polysaccharide is serotype 6B, and wherein the percent ratioof protein/PS is less than 4% and the percent ratio of NA/PS is lessthan 1%.
 16. A solution according to claim 7, wherein the polysaccharideis serotype 7F, and wherein the percent ratio of protein/PS is less than5% and the percent ratio of NA/PS is less than 2%.
 17. A solutionaccording to claim 7, wherein the polysaccharide is serotype 9V, andwherein the percent ratio of protein/PS is less than 2% and the percentratio of NA/PS is less than 1%.
 18. A solution according to claim 7,wherein the polysaccharide is serotype 14, and wherein the percent ratioof protein/PS is less than 3% and the percent ratio of NA/PS is lessthan 2%.
 19. A solution according to claim 7, wherein the polysaccharideis serotype 18C, and wherein the percent ratio of protein/PS is lessthan 2% and the percent ratio of NA/PS is less than 2%.
 20. AStreptococcus pneumoniae serotype 19A capsular polysaccharide, whereinsaid capsular polysaccharide has a molecular weight of 488,000 daltonsto 525,000 daltons.
 21. A solution containing substantially purifiedStreptococcus pneumoniae serotype 19A capsular polysaccharides accordingto claim
 20. 22. A solution according to claim 7, wherein thepolysaccharide is serotype 19A, and wherein the percent ratio ofprotein/PS is less than 2% and the percent ratio of NA/PS is less than2%.
 23. A solution according to claim 22, wherein said polysaccharidehas a molecular weight of 488,000 daltons to 525,000 daltons.
 24. Asolution according to claim 7, wherein the polysaccharide is serotype19F, and wherein the percent ratio of protein/PS is less than 3% and thepercent ratio of NA/PS is less than 2%.
 25. A solution according toclaim 7, wherein the polysaccharide is serotype 23F, and wherein thepercent ratio of protein/PS is less than 2% and the percent ratio ofNA/PS is less than 2%.
 26. A solution according to claim 1, wherein saidsolution is obtained by a process comprising the steps of: (a) providinga fermentation broth comprising bacterial cells that produce a selectedStreptococcus pneumoniae serotype; (b) lysing the bacterial cells instep (a) with a lytic agent, thereby producing a cell lysate comprisingcell debris, soluble proteins, nucleic acids, and polysaccharides; (c)clarifying the cell lysate of step (b) using centrifugation orfiltration to remove cell debris, thereby producing a clarified celllysate; (d) ultrafiltering and diafiltering the clarified cell lysate ofstep (c) to remove low molecular weight impurities and increasepolysaccharide concentration, thereby producing a retentate; (e)lowering the pH of the retentate of step (d) to less than 4.5 toprecipitate protein and nucleic acids, thereby forming an acidifiedretentate solution; (f) holding the acidified retentate solution formedin step (e) for a time sufficient to allow settling of the precipitate,followed by filtration or centrifugation of the acidified retentatesolution, thereby producing a clarified polysaccharide solution; (g)filtering the clarified polysaccharide solution of step (f) through anactivated carbon filter; (h) ultrafiltering and diafiltering thefiltered solution produced by step (g), thereby producing a concentratedpurified polysaccharide solution; and (i) filtering the concentratedpurified polysaccharide solution produced by step (h) using a sterilefilter.
 27. A solution according to claim 1, wherein said solution isobtained by a process comprising the steps of: (a) providing afermentation broth comprising bacterial cells that produce Streptococcuspneumoniae serotype 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, or 23F; (b)lysing the bacterial cells in step (a) with a lytic agent, therebyproducing a cell lysate comprising cell debris, soluble proteins,nucleic acids, and polysaccharides; (c) clarifying the cell lysate ofstep (b) using centrifugation or filtration to remove cell debris,thereby producing a clarified cell lysate; (d) ultrafiltering anddiafiltering the clarified cell lysate of step (c) at room temperatureat neutral pH in salt free media to remove low molecular weightimpurities and increase polysaccharide concentration, thereby producinga salt free retentate; (e) lowering the pH of the salt free retentate ofstep (d) to less than 4.5 to precipitate protein and nucleic acids,thereby forming an acidified retentate solution; (f) holding theacidified retentate solution formed in step (e) for at least 2 hours atroom temperature to allow settling of the precipitate, followed byfiltration or centrifugation of the acidified retentate solution,thereby producing a clarified polysaccharide solution; (g) filtering theclarified polysaccharide solution of step (f) through an activatedcarbon filter;. (h) ultrafiltering and diafiltering the filteredsolution produced by step (g), thereby producing a concentrated purifiedpolysaccharide solution; and (i) filtering the concentrated purifiedpolysaccharide solution produced by step (h) using a sterile filter. 28.A solution according to claim 21, wherein said solution is obtained by aprocess comprising the steps of: (a) providing a fermentation brothcomprising bacterial cells that produce Streptococcus pneumoniaeserotype 19A; (b) lysing the bacterial cells in step (a) with a lyticagent, thereby producing a cell lysate comprising cell debris, solubleproteins, nucleic acids, and polysaccharides; (c) clarifying the celllysate of step (b) using centrifugation or filtration to remove celldebris, thereby producing a clarified cell lysate; (d) ultrafilteringand diafiltering the clarified cell lysate of step (c) at about 4° C. ata pH of about 6 in sodium phosphate buffer to remove low molecularweight impurities and increase polysaccharide concentration, therebyproducing a retentate; (e) lowering the pH of the retentate of step (d)to less than 4.5 to precipitate protein and nucleic acids, therebyforming an acidified retentate solution; (f) holding the acidifiedretentate solution formed in step (e) for at least 2 hours at about 4°C. to allow settling of the precipitate, followed by filtration orcentrifugation of the acidified retentate solution, thereby producing aclarified polysaccharide solution; (g) adjusting the pH of the clarifiedpolysaccharide solution of step (f) to about 6, thereby producing apH-adjusted clarified polysaccharide solution; (h) filtering thepH-adjusted clarified polysaccharide solution of step (g) through anactivated carbon filter; (i) ultrafiltering and diafiltering thefiltered solution produced by step (h), thereby producing a concentratedpurified polysaccharide solution; and (j) filtering the concentratedpurified polysaccharide solution produced by step (i) using a sterilefilter.